Methods of using enantiomerically pure hydroxylated xanthine compounds

ABSTRACT

There is disclosed compounds and pharmaceutical compositions that are a resolved R or S (preferably R) enantiomer of an ω-1 alcohol of a straight chain alkyl (C 5-8 ) substituted at the 1-position of 3,7-disubstituted xanthine. The inventive compounds are effective in modulating cellular response to external or in situ primary stimuli, as well as to specific modes of administration of such compounds in effective amounts.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a Division of U.S. application Ser. No. 08/307,554, filed Sep.16, 1994, still pending, which is a continuation-in-part of U.S. patentapplication Ser. No. 07/926,665 filed on Aug. 7, 1992, now abandoned,which application was a continuation-in-part of U.S. patent applicationSer. No. 07/846,354 filed on Mar. 4, 1992, now abandoned.

TECHNICAL FIELD OF THE INVENTION

The invention relates to a discovery that an isomer of ahydroxy-substituted xanthine compound is an effective agent to modulatecellular responses to stimuli mediated through a stereo-specificcellular second messenger pathway. More specifically, the inventivecompounds are an R or S (preferably R) enantiomer of an ω-1 alcohol of astraight chain alkyl (C₅₋₈) substituted at the 1-position of3,7-disubstituted xanthine. The inventive compounds are usefulantagonists to control intracellular levels of specific sn-2 unsaturatedphosphatidic acids and corresponding phosphatidic acid-deriveddiacylglycerols which occur in response to cellular proliferativestimuli and mediated through a phosphotidic acid (PA) pathway.

BACKGROUND ART

Pentoxifylline (1-(5-oxohexyl)-3,7-dimethylxanthine), abbreviated PTX,is a xanthine derivative which has seen widespread medical use for theincrease of blood flow. PTX is disclosed in U.S. Pat. Nos. 3,422,307 and3,737,433. Metabolites of PTX were summarized in Davis et al., AppliedEnvironment Microbiol. 48:327, 1984. A metabolite of PTX is1-(5-hydroxyhexyl)-3,7-dimethylxanthine, designated M1 and as a racemicmixture. M1 (racemic mixture) was also disclosed as increasing cerebralblood flow (as opposed to just increasing blood flow) in U.S. Pat. Nos.4,515,795 and 4,576,947. In addition, U.S. Pat. Nos. 4,833,146 and5,039,666 disclose use of shorter chain tertiary alcohol analogs ofxanthine for enhancing cerebral blood flow. In subsequent metabolismstudies, PTX was found to be metabolized to the S enantiomer.

Furthermore, U.S. Pat. No. 4,636,507 describes an ability of PTX and M1(racemic mixture), to stimulate chemotaxis in polymorphonuclearleukocytes in response to a stimulator of chemotaxis. PTX and relatedtertiary alcohol substituted xanthines inhibit activity of certaincytokines to affect chemotaxis (U.S. Pat. No. 4,965,271 and U.S. Pat.No. 5,096,906). Administration of PTX and GM-CSF decrease tumor necrosisfactor (TNF) levels in patients undergoing allogeneic bone marrowtransplant (Bianco et al., Blood 76: Supplement 1 (522A), 1990).Reduction in assayable levels of TNF was accompanied by reduction inbone marrow transplant-related complications. However, in normalvolunteers, TNF levels were higher among PTX recipients. Therefore,elevated levels of TNF are not the primary cause of such complications.

It is common practice to market a drug with a chiral center as aracemate. The M1 metabolite has only been disclosed exclusive of itschirality. In fact, M1 appears to be made (metabolically in humans).only as the S isomer. The approach of manufacturing and dosing drugs asracemic mixtures means that each dose of a drug is contaminated with anequal weight of an isomer, which usually has no therapeutic value andhas the potential to cause unsuspected side effects. For example, thesedative thalidomide was marketed as a racemate. The desired sedativeactivity resided in the R-isomer, but the contaminating S-isomer is ateratogen, causing the birth defects in babies born to mothers usingthis drug. The R,R-enantiomer of the tuberculostatic ethambutol cancause blindness. The lethal side effects associated with thenonsteroidal anti inflammatory drug benoxaprofen (Oraflex) might havebeen avoided had the drug been sold as a pure enantiomer.

The issue of enantiomeric purity is not limited to the field ofpharmaceuticals. For example, ASANA (^(i) Pr=isopropyl) is a syntheticpyrethroid insecticide which contains two asymetric centers. The potentinsecticidal activity resides overwhelmingly in just one of fourpossible stereoisomers. Moreover, the three non-insecticidalstereoisomers exhibit cytotoxicity toward certain plant species.Therefore, ASANA can only be sold as a single stereoisomer because themixed stereoisomers would not be suitable.

Therefore, there is a need in the art to discover effective therapeuticcompounds that are safe and effective for human or animal administrationand that can maintain cellular homeostasis in the fade of a variety ofinflammatory stimuli, and that are enantiomerically pure to haveactivity residing in a single isomer. The present invention was made ina process of looking for such compounds.

SUMMARY OF THE INVENTION

We have found that the compounds described herein can be used tomaintain homeostasis of a large variety of target cells in response to avariety of inflammatory and proliferative stimuli. In addition, theinventive compounds and pharmaceutical compositions are suitable fornormal routes of therapeutic administration (e.g., oral, topical andparenteral) and permit effective dosages to be provided.

The inventive compounds and pharmaceutical compositions are a resolved Ror S (preferably R) enantiomer of an ω-1 alcohol of a straight chainalkyl (C₅₋₈) substituted at the 1-position of 3,7-disubstitutedxanthine. The inventive compounds are effective in modulating cellularresponse to external or in situ primary stimuli, as well as to specificmodes of administration of such, compounds in effective amounts.

The inventive compounds comprise compounds and pharmaceuticalcompositions having a compound comprising a xanthine core of theformula: ##STR1## wherein R₁ is independently a resolved enantiomer ω-1secondary alcohol-substituted alkyl (C₅₋₈) substantially free of theother enantiomer, and wherein each of R₂ and R₃ is independently alkyl(C₁₋₁₂) optionally containing one or two nonadjacent oxygen atoms inplace of a carbon atom. Preferably R₁ is a C₆ alkyl with the hydroxylgroup as the R enantiomer.

The present invention further provides a pharmaceutical compositioncomprising an inventive compound and a pharmaceutically acceptableexcipient, wherein the pharmaceutical composition is formulated fororal, parenteral or topical administration to a patient.

The present invention further provides a method for treating anindividual having a variety of diseases, wherein the disease ischaracterized by or can be treated by inhibiting an immune response or acellular response to external or in situ primary stimuli, wherein thecellular response is mediated through a specific phospholipid-basedsecond messenger acting adjacent to the inner leaflet of the cellmembrane of a cell. The second messenger pathway is activated inresponse to various noxious or proliferative stimuli characteristic of avariety of disease states and the biochemistry of this second messengerpathway is described herein. More specifically, the invention isdirected to methods to treat or prevent clinical symptoms of variousdisease states or reduce toxicity's of other treatments by inhibitingcellular signaling through the second messenger pathway describedherein. The disease states or treatment-induced toxicity's are selectedfrom the group consisting of proliferation of tumor cells in response toan activated oncogene; hematocytopenia caused by cytoreductive therapiesor caused by an infection of a microbial agent; autoimmune diseasescaused by a T cell response or a B cell response and antibodyproduction; septic shock; resistance of mesenchymal cells to tumornecrosis factor (TNF); disregulation of cell activation or disregulatedcell growth, such as proliferation of smooth muscle cells, endothelialcells, fibroblasts and other cell types in response to growth factors,such as PDGF-AA, BB, FGF, EGF; etc. (i.e., atherosclerosis, restenosis,stroke, and coronary artery disease); human immunodeficiency virusinfection (AIDS and AIDS related complex); proliferation of kidneymesangial cells in response to IL-1, mip-1α, PDGF or FGF resulting invarious inflammatory renal deseases; inflammation; kidney glomerular ortubular toxicity in response to cyclosporin A or amphotericin Btreatment; organ toxicity (e.g., gastrointestinal or pulmonaryepithelial) in response to a cytoreductive therapy (e.g., cytotoxicdrugs or radiation); enhancing antitumor effects of nonalkylatingantitumor agents; allergies in response to inflammatory stimuli (e.g.,TNF, IL-1 and the like) characterized by production of cell surfacemetalloproteases or by degranulation of mast cells and basophils inresponse to IgE, bone diseases caused by overproduction ofosteoclast-activating factor (OAF) by osteoclasts, CNS diseases causedby reduced signal transduction of the neurotransmitters epinephrine andacetylcholine, and combinations thereof.

When a cell is stimulated to express a particular cytokine or toproliferate in response to a proliferative or noxious stimuli, thisprocess is mediated through a specific phospholipid-based secondmessenger signaling pathway. This second messenger pathway produceselevated levels of a subset of phosphatidic acid (PA) containing sn-2non-arachidonate unsaturation that is rapidly convened to itscorresponding diacylglycerol (DAG). The inventive compounds andpharmaceutical compositions specificly inhibit the stereo-specificenzymes involved in this second messenger pathway without affectingother second messenger pathways that are involved in normalhouse-keeping functions of a cell, such as the phosphatidyl inositol(PI) pathway. The result of inhibiting one or several enzymes involvedin the second messenger pathway described herein is "modulation" of theresponse of a target cell to a stimulus, particulary a noxious stimulus.This biochemical event (i.e., inhibiting activity of a second messengerpathway enzyme that responds to a primary stimulus, such as a cytokine)effects cellular signaling and results in an effect upon many diversedisease states that are the result of abnormal, inflammatory or noxiouscellular signaling mechanisms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a mixed lymphocyte reaction of CT1501R(R-(-)(5-hydroxyhexyl)theobromine) and PTX. The mixed lymphocytereaction shows a proliferative response of PBMC (peripheral bloodmononuclear cells) to allogeneic stimulation determined in a two-waymixed lymphocyte reaction. Both CT1501R and PTX showed dose-responseactivity in this immune modulating activity assay procedure.

FIG. 2 shows the effects of CT1501R on inhibition of murine B-cellproliferation stimulated by anti-mu antibody crosslinked and/orinterleukin-4 (IL-4). FIG. 2 shows that CT1501R inhibited B-cellproliferation caused by the indicated proliferative signals.

FIG. 3 shows the effects of CT1501R inhibiting proliferation caused byConcanavalin A (ConA) and interleukin-1 alpha (IL-1α) or interleukin-2(IL-2). CT1501R was added to the cells at the doses indicated two hoursprior to activation with ConA and IL-1α or IL-2. CT1501R inhibitedthymocyte proliferation in a dose-response manner as is shown in FIG. 3.Background counts were less than 200 cpm.

FIG. 4 shows the effects of CT1501R and PTX on inhibition of smoothmuscle proliferation stimulated by PDGF (platelet derived growth factor)and IL-1. CT1501R and PTX were separately added to the cells two hoursprior to activation with PDGF and IL-1. Both drugs inhibited smoothmuscle cell proliferation at the higher doses tested as shown in FIG. 4.CT1501R was more active than PTX.

FIG. 5 shows inhibition of endotoxin induced lethality in mice byCT1501R as a cumulative percent survival of mice from up to 6independent experiments (N=the number of experiments). Animals weretreated with 10 μg/gm of LPS (i.v.) Immediately following the LPS (t=0),2 hrs after the LPS (t=2) or 4 hrs following LPS (t=4) the mice weretreated with their first treatment of CT1501R (100 mg/kg, i.p.). Themice were treated with CT1501R three times per day thereafter, andanimal survival was monitored.

FIG. 6 shows mouse serum TNF-α levels following endotoxin treatment.Each point represents the average of two experiments compiled from theaverage of duplicate ELISA measurements with each experimental pointrepresenting pooled plasma from three mice.

FIG. 7 illustrates data from mouse plasma IL-1α ELISA measurements. Datawere compiled from an average of duplicate ELISA measurements from asingle experiment with each point representing pooled serum from threemice.

FIG. 8 illustrates data from mouse plasma IL-6 ELISA measurements. Dataare compiled from the average of duplicate ELISA measurements from asingle experiment with each point representing pooled plasma from threemice.

FIG. 9 shows a flow diagram of a large scale synthesis procedure forCT1501R.

FIG. 10 shows the mean WBC of mice treated with 5-fluorouracil on day 0and CT1501R or vehicle control twice daily starting on day -1. Thisexperiment, reported in FIGS. 10-13, is an in vivo model forhematopoiesis. Groups of 4 mice were phlebotomized at each time point.The values on the graph represent the means ±1 SD.

FIG. 11 shows the mean platelet counts of mice treated with5-fluorouracil on day 0 and CT1501R or vehicle control twice dailystarting on day 0. Groups of 4 mice were phlebotomized at each timepoint. The values on the group represent the means ±1 SD.

FIG. 12 shows the mean absolute neutrophil counts of mice treated withCT1501R or vehicle control twice daily starting on day 0. Groups of 4mice were phlebotomized at each time point. The values on the graphrepresent the means ±1 SD.

FIG. 13 shows the mean CFU-GM/femur of mice treated with 5-fluorouracilon day 0 and CT1501R or vehicle control twice daily starting on day 0.Groups of 4 mice were sacrificed and had one femur harvested at eachtime point. The values on the graph represent the means ±1 SD of themean number of colonies per each individual tested in triplicatecultures.

FIG. 14 shows the mouse survival data in the experiment described inFIG. 13 in the form of a Kaplan-Meier plot showing improved survivalwith CT-1501R.

FIGS. 15 and 16 show a comparison of CT1501R with published results fromanother septic shock drug, the bifunctional TNF receptor made byGenentech (Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535, 1991).Both drugs were administered approximately simultaneously (FIG. 15) ortwo hours following (FIG. 16) a lethal LPS (endotoxin) administration.In this series of comparative in vivo experiments, CT1501R consistentlywas better at improving survival than Genentech's TNF receptorirrespective of the time of drug administration.

FIG. 17 illustrates a comparison of CT1501R, its S isomer CT1501S, andPTX on in vitro inhibition of lysophosphatidic acid acyl transferase(LPAAT), an enzyme involved in second messenger signaling. As can beseen, only CT1501R significantly inhibited enzyme activity at IC50concentrations that would be achievable in a clinical setting. LPAATactivity was determined in 3T3 Ras-transformed fibroblasts stimulatedwith IL-1β.

FIG. 18 shows a dose response for inhibition of TNF-α release fromLPS-activated mouse peritoneal macrophages by CT1501R. Cells wereisolated and treated with or without various concentrations of CT1501Rone hour prior to LPS activation (10 mg/ml). Twenty four hours later thesupernatants were collected assayed for TNF-α by ELISA (+/-SD).

FIG. 19 shows inhibition of IL-1-α release in LPS activated PEC byCT1501R. Cells were isolated and treated with or without 250 μM CT1501Rone hour prior to activation with 10 μg/ml LPS. At various limes later,supernatants were harvested and assayed for IL-1-α by ELISA (+/-SD)

FIG. 20 shows inhibition of TNF-α release from IL-1-α-activated mouseperitoneal macrophages. Cells were isolated and treated with or withoutCT1501R (250 μM) 1 hour prior to IL-1-α activation. The supernatantswere collected at 24, 48 or 72 hrs following activation and assayed forTNF-α by ELISA (+/-SD).

FIG. 21 shows inhibition of adhesion of U937 cells to activated HUVEC.HUVEC were stimulated with either IL-1-α (100 ng/ml); TNF-α (10 ng/ml)or LPS (10 mg/ml) with or without the addition of 250 μM CT1501R onehour prior to addition of the stimulus. Twelve hours later, the cellswere assayed for adhesion of BCECF-labeled U937 cells. FIG. 21 is therelative BCECF fluorescence (+/-SD) for each of the treatment groups.

FIG. 22 shows inhibition of adhesion of activated U937 cells to HUVEC.U937 cells were stimulated with various concentrations of IL-1-α with orwithout the addition of 250 μM CT1501R one hour prior to addition ofIL-1-α. Twelve hours later, the U937 cells were stained with BCECF andallowed to adhere to HUVEC. FIG. 22 is the relative BCECF fluorescence(+/-SD) for each of the treatment groups:

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to a defined genus of inventive compoundswhich can control cellular behavior by a particular phase of a secondarymessenger pathway system (Bursten et al., J. Biol. Chem. 266:20732,1991). The second messengers are lipids or phospholipids and use thefollowing abbreviations:

PE=phosphatidyl ethanolamine

LPE=lysophosphoethanolamine

PA=phosphatidic acid

LPA=lysophosphatidic acid

DAG=diacylglycerol

LPLD=lysophospholipase-D

LPAAT=lysophosphatidic acid acyl transferase

PAPH=phosphatidic acid phosphohydrolase

PLA2=phospholipase A-2.

PLD=phospholipase D

PAA=phosphoarachidonic acid

PLA-2=phospholipase A2

PC=phosphatidyl choline

"remodeled" PA, cyclic pathway=PAA, LPA, PA and DAG intermediatessubstituted with L-saturated, 2-linoleoyl or1,2-dileolyl/1,2-sn-dilinoleoyl at the indicated sn-1 and sn-2positions.

"Classical PI Pathway"=PI, DAG, PA intermediates substituted with1-stearoyl, 2-arachidonoyl fatty acyl side chains.

"PLD-generated PA"=PE, PC, LPA, PA and DAG intermediates substitutedwith, e.g., 1,2-sn-dioleoyl-, 1-alkyl, 2-linoleoyl-, and 1-alkyl,2-docosahexaneoyl-side chains.

Lysophosphatidic acid transferase (LPAAT) effects the synthesis ofphosphatidic acid (PA) from lysophosphatidic acid (LPA) by incorporationof an acyl group from acyl CoA. Hydrolysis of the phosphate moiety by PAphosphohydrolase (PAPH) results in the formation of DAG. These aspectsof the pathway appear to be activated immediately (within a minute) uponstimulation by a primary stimulus (e.g., a cytokine such as IL-1, IL-2or TNF) acting at a receptor on a cellular surface. An immediatedetectable effect is an elevation of levels of PA and DAG.Administration of the compounds of the invention reverse this elevation.

The compounds and pharmaceutical compositions of the invention includeinhibitors of subspecies of LPAAT in PAPH enzymes with substratespecificity for intermediates with 1,2-diunsaturated and 1-alkyl,2-unsaturated subspecies. One representative example of such aninhibitor (although not within the genus of inventive compounds) is PTX.PTX blocks PAPH in a specific activation pathway that does not involvePI but rather derives from a PA that is largely composed of1,2-diunsaturated and 1-alkyl,2-unsaturated subspecies. This was shown,for example, by the demonstration that human mesangial cells stimulatedwith TNF produce DAG from PI and regenerate PI in the absence and thepresence of PTX. In the latter system there is no evidence to suggestthat PA or DAG are derived from sources other than PI. It should beemphasized that the compounds of the invention affect that subset ofPAPH and LPAAT that relates to substrates with unsaturated fatty acidsother than arachidonate in the sn-2 position, not the housekeeping formsof these enzymes that serve the PI pathway.

Each membrane phospholipid subclass (e.g., PA, PI, PE, PC and PS)reaches a stable content of characteristic fatty acyl side chains due tocyclic remodeling of the plasma membrane as well as turnover for eachsubclass. PA is often stable, but present in relatively smallquantities. PA in resting cells Consists mostly of saturated acylchains, usually consisting of myristate, stearate and palmitate. Inresting cells, PC's acyl side chains consist mostly of acyl palmitate inthe sn-1 position and oleate in the in-2 position. PE and PI arepredominantly composed of sn-1 stearate and sn-2 arachidonate.

Due to this characteristic content of acyl groups in the sn-1 and sn-2positions, the origin of any PA species may be deduced from the chemicalnature of its acyl groups in the sn-1 and sn-2 positions. For example,if PA is derived from PC through action of the enzyme PLD, the PA willcontain the characteristic acyl side chains of PC substrate passedthrough the second messenger pathway. Further, the origin of any 1,2sn-substrate species may be differentiated as to its origin. However, itis important to know whether or not each phospholipid species passesthrough a PA form previous to hydrolysis to DAG. The lyso-PA that isconverted to PA and thence to DAG may be shown. The complexities of thissecond messenger pathway can be sorted by suitable analyses by fattyacyl side chain chemistry (i.e., by thin layer chromatography or highpressure liquid chromatography) of intermediates in cells at varioustime points after stimulation of the second messenger pathway.

In certain mesengial cells, such as neutrophils and rat or humanmesengial cells, several signaling pathways may be activated in tandem,simultaneously or both. For example, in neutrophils, F-Met-Leu-Phestimulates formation of PA through the action of PLD, followed in timeby formation of DAG through the action of PAPH. Several minutes later,DAG is generated from PI through the classical phosphoinositide pathwayin many cells, DAG is derived from both PA that is being remodeledthrough a cycle whereby PAA is sn-2 hydrolyzed by PLA-2, followed bysn-2 transacylation by LPAAT, and a PLD-pathway from PA that isgenerated from either PE or PC or both substrates by PLD.

A method described here permits differentiation of the varioussubspecies of PA and DAG based upon acyl chain composition. This candifferentiate those compounds that activate (and inhibit activation of)the present second messenger pathway from other pathways, such as theclassical PI pathway. The present second messenger pathway involvessubstrates with unsaturated fatty acids in the sn-2 position other thanarachidonate and those sub species of PAPH and LPAAT that are notinvolved in normal cellular housekeeping functions that are part of theclassical PI pathway. The PAPH and LPAAT enzymes involved in the presentsecond messenger pathway are exquisitely stereo specific for differentacyl side chains and isomeric forms of substrates. Therefore, theinventive compounds are substantially enantiomerically pure, andpreferably are the R enantiomer at the chiral carbon atom bonded to thehydroxyl group. For example, the R and S isomers of CT1501 havedifferent LPAAT inhibiting activity as shown in FIG. 12. Further, the Renantiomer of CT1501 (e.g., CT1501R) is two to three time more potentthan the racemic mixture (designated M1 herein), and many times morepotent than the corresponding S enantiomer. Moreover, PTX is convertedalmost exclusively to the S enantiomer of MI when metabolized in humansor CT1501S.

PTX (in vitro) blocks formation of remodeled PA through the PA/DAGpathway at high PTX concentrations (greater than those that could beachieved in patients without dose-limiting side effects) by blockingformation of PA subspecies at LPAAT. Even in the presence of PTX, cellscontinue to form PA through the action of PLD, and DAG is also formedthrough the action of phospholipase C on PC and PI. The latter pathwayare not inhibited by the inventive compounds or PTX. In PTX-treatedcells, DAG derived from remodeled and PLA-generated PA is diminished(e.g., 1,2-sn-dioleoyl DAG, 1-alkyl,2-linoleoyl DAG and1-alkyl,2-docosahexaneolyl DAG). Therefore, the inventive compounds andPTX inhibit the formation of only a certain species of PA and DAG byselectively inhibiting a specific second messenger pathway that is onlyactivated in cells by noxious stimuli, but is not used to signal normalcellular housekeeping functions.

Therapeutic Uses of the Inventive Compounds

The specific inhibition of activation of the specific second messengerpathway that is activated primarily by various noxious stimuli, providesthe inventive compounds with an ability to be used to treat a widevariety of clinical indications. Moreover, the in vitro and in vivo datapresented herein provides predictive data of a wide variety of clinicalindications that share a common thread of activation of the specificsecond messenger pathway; whose activation by noxious stimuli mediatedthrough, for expame, inflammatory cytokines, is specifically inhibitedby the inventive compounds. In fact, it is this mechanism of action ofthe inventive compounds that explains why the inventive compounds canhave a wide variety of different clinical indications. Activation of thepresent second messenger pathway is a major mediator of response tonoxious stimuli and results in cellular signals that lead to, forexample, inflammation, immune response, inhibition of blood cellregeneration and cancer cell growth. However, not all inhibitors inhibitall enzymes of this second messenger pathway. The inventive compoundsare most effective mediators of inflammation and inhibition of bloodcell regeneration. Signals mediated by the present second messengerpathway include, for example, those cellular responses of LPS directly,T cell activation by antigen, B cell activation by antigen, cellularresponses to IL-1 mediated through the IL-1 Type 1 receptor (but not theIL-1 Type 2 receptor), the TNF Type 1 receptor, activated oncogenes(e.g., ras, abl, her2-neu and the like), low affinity GM-CSF(granulocyte macrophage colony stimulating factor) receptor, and smoothmuscle cell proliferation stimulated by PDGF, b-FGF and IL-1. There areother signals that are not mediated through the present second messengerpathway, and these include proliferation of hematopoietic cells inducedby G-CSF (granulocyte colony stimulating factor), interleukin-3 (IL-3),SCF (stem cell factor) and GM-CSF; neutrophil activation induced byinterleukin-8 (IL-8) or leukotriene B4; T cell proliferation in responseto IL-2; and endothelial cell proliferation in response to acidic FGF(fibroblast growth factor).

In vitro, the inventive compounds: (1) block IL-1 signal transductionthrough the Type 1 receptor as shown, for example, by preventing IL-1and IL-1 plus PDGF (platelet derived growth factor) induction ofproliferation of smooth muscle and kidney mesengial cells; (2)suppresses up regulation of adhesion molecules as shown, for example; byblocking VCAM in endothelial cells of CD18 in neutrophils; (3)inhibiting TNF and IL-1 induced metalloproteases (an inflammationmodel); (4) block LPS-induced cellular activation (for prevention andtreatment of septic shock); (5) suppress T cell and B cell activation byantigen of by cross-linking CD3 complex; (6) inhibit mast cellactivation by IgE; and (7) suppress malignant phenotype in transformedcells and tumor cell lines.

The foregoing in vitro effects give rise to the following in vivobiologic effects, including, but not limited to, protection andtreatment of endotoxic shock, inhibition of tumor cell growth,stimulation of hematopoiesis following cytoreductive therapy,synergistic immunosuppression in preventing GVHD (graft versus hostdisease), and stimulation of hair grow through reversal of an apoptoticprocess. There is in vivo data presented herein showing treatment andprotection of endotoxic shock, stimulation of hematopoiesis followingcytoreductive therapy and stimulation of hair growth in a nude mousemodel. The inventive compounds, and particularly CT1501R, are mostpotent when used to stimulate hematopoiesis, prevent and treat septicshock and stimulate hair growth when applied topically. The inventivecompounds are less potent and require higher molar concentrations forimmunosuppression, acute inflammation, chronic inflammation and GVHD,although inhibitors of the present second messenger pathway can havesuch activities.

The inventive compounds also are useful as an adjuvant to inhibit toxicside effects of drugs whose side effects are mediated through thepresent second messenger pathway. These side effects include, forexample, side effects of interleukin-2 (IL-2), renal side effects ofcyclosporin A and FK506, and side effects of amphotericin B. It shouldbe noted that the inventive compounds inhibit antigen-induced T cellactivation, like cyclosporin of FK506, but, unlike cyclosporin or FK506,do not prevent generation of NK and LAK cells, do not suppress IL-2release from T cells and do not suppress IL-8 release.

Metalloproteases mediate tissue damage such as glomerular diseases ofthe kidney, joint destruction in arthritis, and lung destruction inemphysema, and play a role in tumor metastases. Three examples ofmetalloproteases include a 92 kD type V gelatinase induced by TNF, IL-1and PDGF plus bFGF, a 72 kD type IV collagenase that is usuallyconstitutive and induced by TNF or IL-1, and a stromelysin/PUMP-1induced by TNF and IL-1. The inventive compounds can inhibit TNF or IL-1induction of the 92 kD type V gelatinase inducable metalloprotease.Moreover, CT1501R reduced PUMP-1 activity induced by 100 U/ml of IL-1 to15% of its control levels. PTX, by contrast in the same experiment, onlyinhibited PUMP-1. activity to 95% of its control levels which was notsignificant. Accordingly, the inventive compounds prevent induction ofcertain metalloproteases induced by IL-1 or TNF and are not involvedwith constitutively produced proteases (e.g., 72 kD type IV collagenase)involved in normal tissue remodeling.

The inventive compounds inhibit signal transduction mediated through theType I IL-1 receptor, and are therefore considered as IL-1 antagonists.A recent review article entitled "The Role of Interleukin-1 in Disease"(Dinarello and Wolff N. Engl. J. Med. 328, 106, Jan. 14, 1993) describedthe role of IL-1 as "an important rapid and direct determinant ofdisease." "in septic shock, for example, IL-1 acts directly on the bloodvessels to induce vasodilatation through the rapid production ofplatelet activating factor and nitric oxide, whereas in autoimmunedisease it acts by stimulating other cells to produce cytokines orenzymes that then act on the target tissue." The article describes agroup of diseases that are mediated by IL-1, including sepsis syndrome,rheumatoid arthritis, inflammatory bowel disease, acute and myelogenousleukemia, insulin-dependent diabetes mellitus, atherosclerosis and otherdiseases including transplant rejection, graft versus host disease(GVHD), psoriasis, asthma, osteoporosis, periodontal disease, autoimmunethyroiditis, alcoholic hepatitis, premature labor secondary to uterineinfection and even sleep disorders. Since the inventive compoundsinhibit cellular signaling through the IL-1 Type I receptor and are IL-1antagonists, the inventive compounds are useful for treating all of theabove-mentioned diseases.

For example, for sepsis syndrome: "the mechanism of IL-1-induced shockappears to be the ability of IL-1 to increase the plasma concentrationsof small mediator molecules such as platelet activating factor,prostaglandin and nitric oxide. These substances are potent vasodilatorsand induce shock in laboratory animals. Blocking the action of IL-1prevents the synthesis and release of these mediators. In animals, asingle intravenous injection of IL-1 decreases mean arterial pressure,lowers systemic vascular resistance, and induces leukopenia andthrombocytopenia. In humans, the intravenous administration of IL-1 alsorapidly decreases blood pressure, and doses of 300 ng or more perkilogram of body weight may cause severe hypotension." "The therapeuticadvantage of blocking the action of IL-1 resides in preventing itsdeleterious biologic effects without interfering with the production ofmolecules that have a role in homeostasis." The present inventivecompounds address the need identified by Dr. Denarello by inhibitingcellular signaling only through the IL-1 Type I receptor and not throughthe IL-1 Type II receptor.

With regard to rheumatoid arthritis, Dr. Denarello states:"Interleukin-1 is present in synovial lining and synovial fluid ofpatients with rheumatoid arthritis, and explants of synovial tissue fromsuch patients produce IL-1 in vitro. Intraarticular injections ofinterleukin-1 induce leukocyte infiltration, cartilage breakdown, andperiarticular bone remodeling in animals. In isolated cartilage and bonecells in vitro, interleukin-1 triggers the expression of genes forcollagenases as well as phospholipases and cyclooxygenase, and blockingits action reduces bacterial-cell-wall-induced arthritis in rats."Therefore, the inventive compounds, as IL-1 antagonists, are useful totreat and prevent rheumatoid arthritis.

With regard to inflammatory bowel disease, ulcerative colitis andCrohn's disease are characterized by infiltrative lesions of the bowelthat contain activated neutrophils and macrophages. IL-1 can stimulateproduction of inflammatory eicosanoids such as prostaglandin E₂ (PGE₂)and leukotriene B₄ (LTB₄) and IL-8, an inflammatory cytokine withneutrophil chemoattractant and neutrophil-stimulating properties. Tissueconcentrations of PGE2 and LTB4 correlate with the severity of diseasein patients with ulcerative colitis, and tissue concentrations of IL-1and IL-8 are high in patients with inflammatory bowel disease.Therefore, an IL-1 antagonist, such as the inventive compounds, would beeffective to treat inflammatory bowel disease.

With regard to acute and chronic myelogenous leukemia, there isincreasing evidence that IL-1 acts as a growth factor for such tumorcells. Therefore, the inventive compounds should be effective to preventthe growth of worsening of disease for acute and chronic myelogenousleukemias.

Insulin-dependent diabetes mellitus (IDDM) is considered to be anautoimmune disease with destruction of beta cells in the islets ofLangerhans mediated by immunocompetent cells. Islets of animals withspontaneously occurring IDDM (e.g., BB rats or NOD mice) haveinflammatory cells that contain IL-1. Therefore, the inventive compoundsshould be useful for the prevention of and treatment of IDDM.

IL-1 also plays a role in the development of atherosclerosis.Endothelial cells are a target of IL-1. IL-1 stimulates proliferation ofvascular smooth muscle cells. Foam cells isolated from fatty arterialplaques from hypercholesterolemic rabbits contain IL-1β and IL-1βmessenger RNA. The uptake of peripheral blood monocytes results ininitiation of IL-1 production by these cells. IL-1 also stimulatesproduction of PDGF. Taken together, IL-1 plays a part in the developmentof atherosclerotic lesions. Therefore, an IL-1 antagonist, such as theinventive compounds should be useful in preventing and treatingatherosclerosis.

In Vitro Assays for Physiologic and Pharmacologic Effects of theInventive Compounds

Various in vitro assays can be used to measure effects of the inventivecompounds to module immune activity and have antitumor activity using avariety of cellular types. For example, a mixed lymphocyte reaction(MLR) provides a valuable screening tool to determine biologicalactivity of each inventive compound. In the MLR, PBMCs (peripheral bloodmononuclear cells) are obtained by drawing whole blood from healthyvolunteers in a heparinized container and diluted with an equal volumeof hanks balanced salt solution (HBSS). This mixture is layered on asucrose density gradient, such as a Ficoll-Hypaque® gradient (specificgravity 1.08), and centrifuged at 1000×g for 25 minutes at roomtemperature or cooler. PBMC are obtained from a band at a plasma-Ficollinterface, separated and washed at least twice in a saline solution,such as HBSS. Contaminating red cells are lysed, such as by ACK lysisfor 10 min at 37° C., and the PBMCs are washed twice in HBSS. The pelletof purified PBMCs is resuspended in complete medium, such as RPMI 1640plus 20% human inactivated serum. Proliferative response of PBMC toallogeneic stimulation is determined in a two-way MLR performed in a96-well microtiter plate. Briefly, approximately 10⁵ test purified PBMCcells in 200 μl complete medium are co-cultured with approximately 10⁵autologous (control culture) or allogeneic (stimulated culture) PBMCcells, wherein the allogeneic cells are from HLA disparate individuals.Varying doses of compounds (drug) are added at the time of addition ofcells to the microtiter plate. The cultures are incubated for 6 days at37° C. in a 5% CO₂ atmosphere. At the conclusion of the incubationtritiated thymidine is added (for example, 1 μCi/well of 40 to 60Ci/mmole). and proliferation determined by liquid scintillationcounting.

A thymocyte costimulator assay is conducted to evaluate the inventivecompounds to inhibit activation and proliferation of thymocytes causedby stimulation with Con A and interleukin-1 (IL-1), or interleukin-1(IL-2). Thymuses are obtained from mice (e.g., female Balb/C mice) andthe thymuses are removed and dissociated into culture media (e.g., RPMI1640 without serum supplementation). The dissociated thymus tissue andcell suspension is transferred to centrifuge tubes and allowed tosettle, washed with HBSS and resuspended in serum-supplemented culturemedia (e.g., RPMI 1640 with 10% fetal calf serum). Any contaminating redcells are lysed, and viable cells are resuspended and counted.Thymocytes are plated (e.g., 96-well plates at a density of 2×10⁵cells/well) and a stimulating agent, such as Con A, IL-1 (e.g., IL-1α)or IL-2 is added to the well. The cells are incubated for 4 days at 37°C. On the fourth day, the cells are pulsed with tritiated thymidine andcell proliferation determined. Inventive compounds are added at the timeof stimulating agent addition.

Each inventive compound is investigated for cytotoxicity to determineappropriate doses for biological activity assays and to preventcytotoxic reactions in in vitro assays when characterizing activity.Cells (e.g., NH-3T3, Ras transformed 3T3 cells, malignant melanoma LD2cells, etc.) are added to microtiter plates and drug is added about twodays after plating. Cell viability is determined using a fluorescentviability stain (e.g., 2',7'-bis-(2-carboroxyethyl)-5(and-6)-carboxyfluorescein acetoxymethyI ester, BCECF excitation 488 nm andemission 525 nm) 24, 48 or 72 hours after addition of the drug.

Another assay for measuring activity of the inventive compounds involvesdetermining PDGF (platelet derived growth factor) proliferative responseusing human-derived stromal cells. Human stromal cells are plated (e.g.,about 2000 cells per well) in defined media (e.g., 69% McCoy's, 12.5%fetal calf serum, 12.5% horse serum, 1% antibiotics, 1% glutamine, 1%vitamin supplement, 0.8% essential amino acids, 1% sodium pyruvate, 1%sodium bicarbonate, 0.4% non-essential amino acids and 0.36%hydrocortisone). Two to three days later, the stromal cells are starvedin serum-free media. Twenty four hours later, the cells are treated witha stimulating agent, such as PDGF-AA, PDGF-BB or basic FGF (fibroblastgrowth factor) with or without IL-1α or TNF, and tritiated thymidine.Cell proliferation is determined by liquid scintillation counting.

A B-cell proliferation assay determines the effect of the inventivecompounds on inhibiting proliferation of stimulated B-cells, stimulatedby an anti-mu antibody (40 μg/ml), IL-4 or PMA (2.5 nM). Ramos B-celltumor cells or murine splenocytes can be incubated with a stimulatingagent, an inventive compound and tritiated thymidine to measureinhibition of cell proliferation caused by the stimulating agent.

Drug inhibitory activity can also be measured by determining levels ofvascular cell adhesion molecule (VCAM) in stimulated cells. Earlypassage human umbilical vein endothelial cells (HUVEC) (obtained fromcommercial suppliers such as Cell Systems, Inc. or Clonetics) arecultured in media (e.g., Hepes buffered media, Cell Systems) containing10% fetal bovine serum, and supplemented with a stimulating agent, suchas fibroblast growth factor (acidic FGF, Cell Systems, Inc.) or TNF. Thecells are plated into wells of a microtiter plate (e.g., 5×10⁴ per well)and allowed to incubate at 37° C. for 72 hrs. The resting cells areremoved (e.g., 20-30 min treatment with 0.4% EDTA), washed in media(e.g., phosphate buffered saline plus 0.1% bovine serum albumin with0.01% sodium azide) and labeled on ice with a monoclonal antibody("first antibody") recognizing human VCAM (e.g., 1 μg of a murinemonoclonal antibody recognizing human VCAM, Genzyme). After 60 min onice, the cells are washed (preferably twice) with cold wash media andincubated with an antibody that recognizes the first antibody (e.g., 1μg of goat anti-mouse IgG Conjugated with phycoerythrin, CalTag, Inc.).After 30 min on ice, the cells are washed twice and analyzed on a flowcytometer (Coulter Elite®) at appropriate emission and excitationwavelengths (e.g., for phycoerythrin use excitation at 488 nm andemission at 525 nm).

One in vitro assay measures inhibition of the relevant enzymeslysophosphatidic acid acyltransferase (LPAAT) and phosphatidic acidphosphoryl hydrolase (PAPH). The assay involves incubating of targetcells with a primary stimulus (e.g., a variety of cytokines, growthfactors, oncogene products, putative therapeutic agents, irradiation,viral infection, toxins, bacterial infection and the products thereof,and any stimulus which, if not counteracted, has a deleterious effect onthe target cell) in the presence or absence of an inventive compound atvarying dosage levels. Target cells include, for example, subcellularentities, such as, microsomes derived from mesenchymal and/or ectodermalcells, particularly microsomes from marrow stromal cells or human or ratmesangial cells; microsomes or synaptosomes derived from brain; plasmamembrane-enriched microsomes, plasma membranes derived as described inBursten et al. (J. Biol. Chem. 226:20732-20743, 1991), ordetergent-solubilized microsomes; synaptosomes, and membranes or othercell preparations solubilized using, for example, NP40, Miranal, SDS orother neutral detergents; and detergent-solubilized, recombinant, orfurther purified preparations of cell proteins, including the proteinsLPAAT and/or PAPH. After incubation for short periods of time, celllipids are extracted and assayed by thin layer chromatography accordingto standard procedures. Briefly, lipids are extracted using, forexample, chloroform:methanol 2:1 (v/v), and the extracts are thensubjected to HPLC as described in Bursten and Harris, Biochemistry30:6195-6203, 1991. A Rainin® mu-Porasil column is used with a 3:4hexane:propanol organic carrier and a 1-10% water gradient during thefirst 10 minutes of separation. Detection of the peaks in the elutionpattern is by absorption in the range of ultraviolet which detectsisolated double bonds. The relevant peaks of unsaturated PA and DAG areshown in the elution pattern. It is important to note that the assaymethod permits discrimination between various forms of PA and DAG sothat those relevant to the pathway affected by the (R) or (S) compoundsof the invention can be measured directly. Confirmation of the nature ofthe acyl substituents of these components is accomplished usingfast-atom bombardment mass spectroscopy. Thus, the relevant unsaturated(non-arachidonic) PA and DAG subspecies may be detected. The timeperiods employed are 5-60 seconds after stimulation with the primarystimulus, such as a cytokine. This technique permits assessment of thelevels of various lipid components as a function of time.

An inventive compound can be assayed for activity protectingTNF-mediated cytotoxicity. In this assay, L929 murine fibroblast cells(10⁴ cells per well) are incubated with either compounds at varyingdoses and media control for two hrs. TNF-α (R&D Systems) is added at aconcentration of 500 pg/ml, which is four times the LD50 of TNF (125pg/ml). The cells plus (or minus) drug, plus TNF were incubated for 40hrs at 37° C. The media is removed and replaced with fresh mediacontaining 2% serum and 10 μg/ml of BCECF fluorescent dye and incubatedfor 30 min. The fluorescent dye-containing media is removed and replacedwith PBS (phosphate buffered saline) and each well was assayed forfluorescence.

Another assay measures the effects of drug to inhibit adhesion of U937cells to TNF-activated HUVEC cells in this experiment, HUVEC cells areinduced with human TNF-α (20 ng/ml) and drug at varying concentrationsfor 14-16 hrs. U937 cells (a human monocyte cell line) are incubated andlabeled with BCECF (10 μg/ml), a fluorescent dye. The U937 cellpreparation (2.5×10⁴ cells per well) is layered on top of the activatedHUVEC cells. The cells are reverse spun to remove partially adhering andnonadhering U937 cell. The adherent U937 cells are measured byfluorescence on a fluorescent plate reader.

Compounds of the Invention

The inventive compounds and pharmaceutical compositions are a resolved Ror S (preferably R) enantiomer of an ω-1 alcohol of a straight chainalkyl (C₅₋₈) substituted at the 1-position of 3,7-disubstitutedxanthine. The inventive compounds are effective in modulating cellularresponse to external or in situ primary stimuli, as well as to specificmodes of administration of such compounds in effective amounts.

The inventive compounds comprise compounds and pharmaceuticalcompositions having a compound comprising a xanthine core of theformula: ##STR2## wherein R₁ is independently a resolved enantiomer ω-1secondary alcohol-substituted alkyl (C₅₋₈) substantially free of theother enantiomer, and wherein each of R₂ and R₃ is independently alkyl(C₁₋₁₂) optionally containing one or two nonadjacent oxygen atoms inplace of a carbon atom. Preferably R₁ is a C₆ alkyl with the hydroxylgroup as the R enantiomer.

The present invention further provides a pharmaceutical compositioncomprising an inventive compound and a pharmaceutically acceptableexcipient, wherein the pharmaceutical composition is formulated fororal, parenteral or topical administration to a patient.

The present invention further comprises a pharmaceutical compositioncomprising one or a plurality of inventive compounds and apharmaceutically acceptable carrier or excipient. The individuals to betreated with an inventive compound or inventive pharmaceuticalcomposition may either be contacted with the compound of the inventionin vitro culture, in an extracorporeal treatment, or by administering(oral, parenteral or topical) the compound of the invention orpharmaceutical composition to a subject whose cells are to be treated.

The (R) or (S) enantiomer of 1-(5-hydroxy-n-hexyl)-3,7-dimethylxanthineare known and prepared by conventional methods known in the artincluding microbial techniques, such as those described in Davis et al.,Xenobiotica, 15:1001, 1985 and Davis et al., Applied and EnvironmentalMicrobiology 48:327, 1984, e.g., using Rhodotorula rubra, resolution ofthe racemate by formation of diastereomeric esters made with opticallyactive acids such as (R)-alpha-methoxy-alpha-trifluormethyl-phenylacetic acid ((r)-MTFPA) andother optically active acids as in Davis et al (1984) supra, directchemical synthesis by introducing the (R) or (S)-S-hydroxy-n-hexylresidue into the 1-position of the dimethylxanthine as described inEuropean patent publication 0 435 153 and enantiomer selectiveconversion, e.g., wherein an enantiomeric ester is heated with atertiary phosphine, an azodicarbonic acid dialkyl ester and a carboxylicacid, or an enantiomeric alcohol is converted into an organic sulfonicacid ester in an aprotic solvent optionally in the presence of base. Theresulting aliphatic carbonic acid ester is heated to achieve solvolysisin the presence of base in an alcoholic or aqueous solvent (e.g.,methanolysis in the presence of potassium carbonate) to obtain theenantiomeric alcohol as described in European patent publication 435 153or 435 152.

A particularly preferred way of obtaining either the (R) or (S)enantiomer is a direct synthesis based on a modification of the methoddisclosed in J. Org. Chem. 55:2274, 1990 and shown as FIG. 9. In thismethod, 4-bromo-1-butene is heated with boron trichloride andtriethylsilane in an inert hydrocarbon solvent such as pentane under lowtemperature conditions of about -10° C. to -78° C. to yield 4-bromo-1(dichloro) borobutane. This product is then treated at about the samelow temperature conditions with (R)- or (S)-pinanediol in an inertsolvent such as diethyl ether or diglyme to give (R)- or (S)-pinanediol4-bromobutylborate. This product is treated in an inert atmosphere suchas argon with LiCHCl₂ at -100° C. (or somewhat warmer) in an inertsolvent such as tetrahydrofuran followed by addition of anhydrous zincchloride to yield (R)- or (S)-pinanediol 5-bromo-1-chlorpentylborate.This product is treated with methyl magnesium bromide at about -10° C.to about -78° C. in tetrahydrofuran to give R or S pinanediol5-bromo-1-methylpentylborate. This product is treated with theobrominein dimethyl sulfoxide (DMSO) at about -10° C. to about 0° C. followed bydistilling off the DMSO at about 28° C. and 5 torr to yield thecorresponding 1-N-alkylated xanthine in very high yield of about 95%.The subsequent oxidative hydrolysis with a strong basic solution, suchas excess aqueous sodium hydroxide, removes the boron atom and (R)- or(S)-pinanediol residue. The products are obtained by conventionalrecovery methods such as recrystallization from a solvent likeisopropanol. The following also illustrates this preferred synthesismethod. ##STR3##

Pinanediol is the chiral director used in the preferred synthesismethod. Pinanediol can be synthesized according to the followingsynthesis procedure.

Outline of the synthesis of pinanediol (chiral director): ##STR4## Thesynthesis procedure utilizes -(-)-α-pinene, which is often not availablein commercial scale quantities. However, -(-)-α-pinene can be made from-(-)-β-pinene according to the following procedure.

Outline of α-pinene synthesis: ##STR5## It is also important toregenerate pinanediol from reaction 10 to recycle this valuable startingmaterial and chiral director to use repeatedly in many reactions. Apreferred recycling method to regenerate pinanediol is illustrated asfollows:

Recovery of pinanediol from Rx 10 for recycling into Rx 7: ##STR6##

Uses of the Invention Compounds and Pharmaceutical Formulations

The compounds of the invention provide a mechanism to maintainhomeostasis in cells contacted by primary stimuli through mitigating theeffects of these primary stimuli on the secondary signaling pathwaysinvoked within seconds of the primary stimulus. For example,administration of the inventive compounds in vivo or ex vivo provide amethod to modify cellular behavior which method comprises contactingcells (in vivo or ex vivo) whose behavior is to be modified with aneffective amount of an inventive compound or a pharmaceuticalcomposition thereof wherein said method is: (I) a method to inhibitproliferation of tumor cells and said amount is sufficient to inhibitsaid proliferation; or (2) a method to promote differentiation ofhematopoietic stem cells into red blood cells, platelets, lymphocytes,and granulocytes, and said amount is sufficient to promote saidproliferation; or (3) a method to suppress activation of T-cells byantigen or IL-2 stimulation, and said amount is sufficient to promotesaid activation; or (4) a method to suppress activation ofmonocyte/macrophage cells by endotoxin, TNF, IL-1 or GM-CSF stimulationand said amount is sufficient to suppress said activation; or (5) amethod to enhance the resistance of mesenchymal cells to the cytotoxiceffect of tumor necrosis factor and said amount is sufficient to enhancesaid resistance; or (6) a method to suppress antibody production ofB-cells in response to an antigen, IL-4 or CD40 ligand and said amountis sufficient to suppress said antibody production; or (7) a method toinhibit the proliferation of smooth muscle cells in response to growthfactors capable of stimulating said proliferation and said amount issufficient to inhibit said proliferation; or (8) a method to lowersystemic vascular resistance conferred by endothelial cells and saidamount is sufficient to reduce the release of hypertension-inducingsubstances; or (9) a method to lower systemic vascular resistanceinduced by endothelial cells and said amount is sufficient to enhancethe release of anti-hypertensive substances; or (10) a method to lowerexpression of adhesion molecules induced by enhancers thereof, and saidamount is sufficient to lower said expression; or (11) a method tosuppress the activation of T-cells by HIV and said amount is sufficientto suppress said activation thus inhibiting viral replication; or (12) amethod to inhibit the proliferation of kidney mesangial cells inresponse to stimulation by IL-1 and/or mip-1α and/or PDGF and/or FGF andsaid amount is sufficient to inhibit said proliferation; or (13) amethod to enhance the resistance of kidney glomerular or tubular cellsto cyclosporin A or amphotericin B and said amount is sufficient toenhance said resistance; or (14) a method to prevent the suppression ofgrowth stimulatory factor production in TNF-treated bone marrow stromalcells and said amount is sufficient to prevent said suppression; or (15)a method to prevent the release of mip-1α by IL-1, TNF, or endotoxinstimulated monocytes and macrophages; or (16) a method to prevent therelease of platelet activating factor by IL-1, TNF, or endotoxin treatedmegakaryocytes, fibroblastic cells, and macrophages; or (17) a method toprevent the down-regulation of receptors for cytokines in TNF-treatedhematopoietic progenitor cells and said amount is sufficient to preventsaid down-regulation; or (18) a method to suppress the production ofmetalloproteases in IL-1-stimulated or TNF-stimulated glomerularepithelial cells or synovial cells and said amount is sufficient toenhance said production; or (19) a method to enhance the resistance ofgastrointestinal or pulmonary epithelial cells to cytotoxic drugs orradiation and said amount is sufficient to enhance said resistance; or(20) a method to enhance the antitumor effect of a non-alkylatingantitumor agent and said amount is sufficient to enhance said effect, or(21) a method to inhibit the production of osteoclast activating factorin response to IL-1, and said amount is sufficient to inhibit saidproduction, or (22) a method to inhibit degranulation in response toIgE, and said amount is sufficient to inhibit said degranulation, or(23) a method to enhance the release of adrenergic neural transmitters,dopamine, norepinephrine, or epinephrine, or the neurotransmitter,acetylcholine, and said amount is sufficient to enhance said release, or(24) a method to modulate the post-synaptic "slow current" effects ofthe adrenergic neurotransmitters dopamine, epinephrine, ornorepinephrine, or the neurotransmitter acetylcholine, and said amountis sufficient to modulate such slow currents.

For example, the compounds of the invention are used in connection withpatients undergoing bone marrow transplantation (BMT), regardless ofwhether the BMT is matched allogeneic, mismatched allogeneic, orautologous. Patients receiving autologous transplants are aided bytreatment with compounds of the invention even though they do notnecessarily need to be administered immunosuppressive agents, since theydo not develop graft-versus-host disease (GVHD). However, the toxiceffect of the chemotherapy or radiation therapy used in connection withthe disease, in response to which the transplantation has beenperformed, constitutes a negative stimulus with regard to the patients'cells.

In general, all patients undergoing BMT require doses of chemotherapywith or without total body irradiation that exceed the lethal dose fornormal bone marrow recovery. This provides the rationale for usingeither stored patient marrow or donor marrow to rescue the patient. Ingeneral, chemotherapy and radiation are delivered to the patient for7-10 consecutive days before the new or stored bone marrow is infused.The day on which the marrow is given to the patient is referred to asday 0 of the transplant. Previous days on which the patient receivedchemo/radiation are designated by negative numbers. Subsequent days arereferred to by positive numerals.

The median time in which negative responses in BMT recipients occurs iswithin the first 100 days after transplant. Therefore, statistically, ifpatients survive through day 100, their chances for continued survivalare significantly enhanced. Inventive compounds are able to increase thepercentage of patients who survive. The percentage of fatalities withinthe first 100 days that is considered acceptable is 15-20% for "goodrisk" patients and 30-40% for "high risk". These fatalities are due tothe direct effects of high doses of chemo/radiation. In addition, GVHDcontributes to the death rate in allogeneic marrow recipients.

Other indications for which it is useful to administer the compounds ofthe invention include the presence of a tumor burden, a hormone-relateddisorder, a neurological disorder, an autoimmune disease/inflammation,restenosis, coronary artery disease, atherosclerosis, hypertension,unwanted immune response, viral infection, nephritis, mucositis, andvarious allergic responses. Prevention of allergic responses includeprevention of acute allergic response and thus moderation or preventionof rhinorrhea, sinus drainage, diffuse tissue edema, and generalizedpruritus. Other symptoms of chronic allergic response include, as wellas the foregoing, dizziness, diarrhea, tissue hyperemia, and lacrimalswelling with localized lymphocyte infiltration. Allergic reactions arealso associated with leukotriene release and the distal effects thereof,including asthmatic symptoms including development of airwayobstruction, a decrease in FEV1, changes in vital capacity, andextensive mucus production.

Other suitable subjects for the administration of compounds of theinvention, include patients being administered cytoreductive agents forthe treatment of tumors, such as chemotherapeutic agents or irradiationtherapy, as well as treatment with biological response modifiers such asIL-2 and tumor suppressing cells such as lymphokine activated killercells (LAK) and tumor-infiltrating lymphocytes (TIL cells); patientssuffering from neoplasias generally, whether or not otherwise treatedincluding acute and chronic myelogenous leukemia, hairy cell leukemia,lymphomas, megakaryocytic leukemia, and the like; disease states causedby bacterial, fungal, protozoal, or viral infection; patients exhibitingunwanted smooth muscle cell proliferation in the form of, for example,restenosis, such as patients undergoing cardiac surgery; patients whoare afflicted with autoimmune diseases, thus requiting deactivation of Tand B cells, and patients who have neurological disorders.

The compounds of the invention further are able to decrease enhancedlevels of a relevant PA and DAG resulting from stimulation ofsynaptosomes with acetylcholine and/or epinephrine. This suggests thatthe effects of the compounds of the invention are to both enhance therelease of inhibitory neural transmitters such as dopamine, and tomodulate the distal "slow current" effects of such neurotransmitters.

Thus, the drugs of the invention are also useful to raise the seizurethreshold, to stabilize synapses against neurotoxins such as strychnine,to potentiate the effect of anti-Parkinson drugs such as L-dopa, topotentiate the effects of soporific compounds, to relieve motiondisorders resulting from administration of tranquilizers, and todiminish or prevent neuron overfiring associated with progressive neuraldeath following cerebral vascular events such as stroke. In addition,the compounds of the invention are useful in the treatment ofnorepinephrine-deficient depression and depressions associated with therelease of endogenous glucocorticoids, to prevent the toxicity to thecentral nervous system of dexamethasone or methylprednisolone, and totreat chronic pain without addiction to the drug. Further, the compoundsof the invention are useful in the treatment of children with learningand attention deficits and generally improve memory in subjects withorganic deficits, including Alzheimer's patients.

While dosage values will vary, therapeutic efficacy is achieved when thecompounds of the invention are administered to a human subject requiringsuch treatment as an effective oral, parenteral, or intravenoussublethal dose of about 200 mg to about 5000 mg per day, depending uponthe weight of the patient. A particularly preferred regimen for use intreating leukemia is 4-50 mg/kg body weight. It is to be understood,however, that for any particular subject, specific dosage regimensshould be adjusted to the individual's need and to the professionaljudgment of the person administering or supervising the administrationof the inventive compounds.

Coadministration With a P-450 Inhibitor

The coadministration in vivo of the compounds of the invention alongwith an inhibitor of P-450 results in an enhanced effect due to a longerhalf life of the inventive compounds. This in vivo effect is due toinhibition of a degradation pathway for the compounds of the invention;in particular, dealkylation at the N7 position of the xanthine ring. Forexample, NIH3T3-D5C3 cells can be used to compare effects of aninventive compound alone or in combination with a P-450 inhibitor bycomparing transformation phenotype control, incubation with an inventivecompound alone, and coincubation of an inventive compound with the P-450enzyme inhibitor.

Compounds that inhibit P-450 include, for example, (mg range dailydosage) propranolol (20-100), metaprolol (20-100); verapamil (100-400),diltiazem (100-400), nifedipine (60-100); cimetidine (400-2,400);ciprofloxacin (500-2000), enoxacin (500-2,000), norfloxacin (500-2000),ofloxacin (500-2,000), pefloxacin (500-2,000); erythromycin (100-1,000),troleandomycin (100-1,000); ketoconizole (100-2,000), thiabenzadole(100-1,000); isoniazid (100-1000); mexiletine (100-1,000); anddexamethasone (1-100 mg).

A suitable formulation will depend on the nature of the disorder to betreated, the nature of the medicament chosen, and the judgment of theattending physician. In general, the inventive compounds are formulatedeither for injection or oral administration, although other modes ofadministration such as transmucosal or transdermal routes may beemployed. Suitable formulations for these compounds can be found, forexample, in Remington's Pharmaceutical Sciences (latest edition), MackPublishing Company, Easton, Pa.

Depending on the inventive compound selected, the level of dosage can beappreciably diminished by coadministration of a P450 inhibitor, such asthe quinolone. Alternatively, a strong synergistic effect may beobtained with such a quinolone.

The invention is illustrated by the following examples which should notbe regarded as limiting the invention in any way. In these examples PTXmeans pentoxifylline.

EXAMPLE 1

This example illustrates a synthesis for CT1501R by resolution ofracemic M1. To ether saturated with M1 in a reaction vial was added 3.0equivalent of pyridine (freshly distilled from calcium hydride) and 3equivalents of the acid chloride ofR-(+)-1-methoxy-1-trifluoromethyl-phenylacetic acid ((+)-MTFPA). Thereaction vial was sealed, 30 warmed at fifty degrees C. for 1 hour,placed under a stream of nitrogen until dry, and then reconstituted in75% MeOH/H₂ O. Separation was achieved using an isocratic system of 90%(75% MeOH/water), 3% acetonitrile (AcN), 7% water at a flow rate of 3.0ml/min through a 250×10 mm Ultremex 5 C-18 column (Phenomenex, Torrance,Calif. 90501). Samples were prepared as 10% solutions of MTFPA-M1 in 75%MeOH/water, 100 μl aliquots were injected every 7 min for 21 min (4injections). Compounds were eluted at 29.8 min (S-M1)-MTFPA, and 31.4min (R-M1 )-MTFPA with 95% separation. Assignment of absoluteconfiguration was based on NMR, as described by Dale et al. (J. Org.Chem. 34:2543, 1969). Collected fractions were combined and reduced involume with a rotovap. Initially, samples were evaporated only to removeMeOH and AcN after which they were extracted with chloroform. Thesolvent was dried and removed in vacuo. Later, the extraction step wasomitted after studies showed that the ester was stable to the minorelevation of temperature required to remove water.

Hydrolysis of (+)-MTFPA-M1 Stereoisomers

For monitoring the progress of hydrolysis of MTFPA esters, it wasnecessary to develop an assay that would quantitate M1 directly as wellas the total ester present. A gradient program was used changing themobile phase from 40% (75% MeOH/water) 10% AcN 50% water to 40% (75%MeOH/water) 60% AcN from 1.0-8.0 min after injection. The finalconditions were maintained for 3 min after which the column wasreequilibrated to starting conditions. Eluent was monitored at 280 nmand retention times were 6.5 min and 12.3 min for free and derivatizedM1, respectively. Using this system there was no separation ofstereoisomers of derivatized M1 and so it could be used as an assay formonitoring of hydrolysis of MTFPA esters.

To 30 mg of pure MTFPA-M1 ester (R or S derivative) in 4.0 ml EtOH wasadded ca. 40 mg sodium borohydride (NaBH) and this mixture was heated inan oil bath to 70° C. Additional sodium borohydride (20 mg) was addedevery 4 hours during the day. After 54 hours the reaction wasapproximately 90% complete (via HPLC) with no loss of the total peakarea of M1+M1-ester. The reaction was terminated with monobasic sodiumphosphate/water, adjusted to pH 3.5 with HCl, the EtOH was removed invacuo, and unreacted ester and M1 extracted into chloroform. Thechloroform was dried over sodium sulfate and removed in vacuo. The crudeproduct was purified using preparative chromatography with the solventgradient outlined above for M1 using a flow rate of 3.0 ml/min with the250×10 mm column. Collected fractions were reduced to dryness in vacuo,triturated with ether, and analyzed for enantiomeric excess (>95% forboth isomers). Ether was removed under nitrogen and the resultingcrystals dissolved in a minimal volume of normal saline. Dilutions weremade of this standardized solution with normal saline to produce 10 mMsolutions of each enantiomer. Enantiomeric identity was confirmed byHPLC of reformed MTFPA esters.

EXAMPLE 2

This example illustrates a process for preparing (R)1-(5-hydroxyhexyl)-3,7-dimethylxanthine) (CT1501R) using R pinanediol asa chiral director on a laboratory scale. Triethylsilane (83.49 g, 0.718moles) and 4-bromo-1 -butene (97 g, 0.718 moles) were mixed in a 500 mlround bottomed flask and cooled to -78° C. In a one liter round bottomedflask, boron trichloride (84.13 g, 0.718 moles) gas was condensed at-78° C. and 400 ml of pentane were added. The silane/butene mixture wasadded to the boron trichloride solution dropwise via a canula withstirring, under argon, while maintaining an internal temperature of -78°C. When the addition was completed, three equivalents of methanol wereadded dropwise to the. The solution was then warmed to room temperature,and the pentane, HCl, and excess methanol were distilled off under argonat atmospheric pressure. The residue was vacuum distilled to givedimethyl 4-bromobutyl boronate; (bp 70°-79° C. at 0.9 torr, yield 127.5g, 85% yield).

In a 500 ml round bottomed flask, (R)-pinanediol (62 g; 0.365 moles) anddimethyl 4-bromobutylboronate (75 g; 0.359 moles) were stirred with 200ml of diethyl ether. After 30 min the ether was removed under vacuum andthe residue was distilled to yield (R)-pinanediol-4-bromobutyl boronate;(bp 134°-141° C. at 1.6-1.9 torr, 110.85 g, 98% yield).

To perform the homologation reaction, methylene chloride (31.47 g; 0.370moles) and 500 ml of anhydrous THF were cooled to -100° C. under argonwith stirring in a one liter round bottomed flask with a side arm. Tothe cooled solution, 212 ml of n-butyllithium (1.4N in hexanes) wereadded dropwise down the side of the flask over 45 min with stirringunder argon while maintaining the internal temperature at -100° C. Thesolution was allowed to stir 20 min after addition was complete.Pinanediol 4-bromobutylboronate (77.82 g; 0.247 moles) was mixed with100 ml anhydrous THF, cooled to -78° C., and then added to the lithiummethyl dichloride solution dropwise while keeping the internaltemperature at 100° C. Upon completion of the addition, rigorously driedzinc chloride (30.29 g; 0.222 moles) was added. The solution was stirredunder argon for 10 hr and warmed to room temperature. The solvents wereremoved under vacuum. To the residue was added 500 ml petroleum etherand 300 ml saturated aqueous ammonium chloride. The organic phase wasseparated and washed with saturated ammonium chloride (2×250 ml). Theaqueous phases were combined and washed with petroleum ether (2×250 ml).The organic phases were combined and dried with sodium sulfate, filteredand evaporated to give crude pinanediol (R) bromopentylboronate, crudewt. 92.13 g (102%).

In a 500 ml round bottomed flask, 300 ml of anhydrous THF and the crudepinanediol (R)-1-chloro-5-bromopentylboronate from the previous reaction(assuming 89 g; 0.247 moles) were mixed and cooled to -78° C. withstirring under argon. To the solution was added methylmagnesium bromide(3.26N, 79.6 ml). The solution was warmed to room temperature overnight.Petroleum ether (500 ml) and saturated ammonium chloride (250 ml) wereadded, forming an emulsion. The aqueous, phases were separated. Theorganic phase was washed with saturated ammonium chloride (2×250 ml),causing the emulsion to disappear. The combined aqueous phases werewashed with petroleum ether (2×250 ml). The combined organic phases weredried over sodium sulfate, filtered and evaporated under vacuum to yield85.85 g of crude pinanediol (R)-5-bromo-1-methylpentylboronate.

In a 5 liter flask, 2 liters of DMSO and theobromine (44.51 g, 0.247moles) were combined with stirring under argon. Sodium hydride (9.9 g;0.247 moles) was added in two aliquots to the solution and allowed tostir until the theobromine was dissolved. After 3 hr, the pinanediol(R)-5-bromo-1-methylpentylboronate from the previous reaction (84.75 g;0.247 moles) was added neat to the solution dropwise and allowed to stirfor 12 hr.

The DMSO was distilled from the solution (it may be recycled). Theresidue was treated with 500 ml of methylene chloride and 500 ml water.The aqueous phase was removed and the organic phase was washed (3×750ml) with water. The aqueous phases were combined and extracted with2×500 ml of methylene chloride. The organic phases were combined, driedwith sodium sulfate, filtered and the solvents removed under vacuum toyield 88.14 g, 80% yield) of pinanediol(R)-5-(3,7-dimethylxanthine)-1-methylpentyl boronate.

The boronate was then dissolved in THF/water (300 ml each) and themixture cooled to 0° C. with stirring under argon. While maintaining theinternal temperature at 0° C., 95 ml of 3N potassium hydroxide was addeddropwise followed by dropwise addition of 32.3 ml of 30% hydrogenperoxide. The mixture was stirred for 2 hr and the solids were filteredoff. Water and methylene chloride (300 ml each) were added. The phaseswere separated and the aqueous phase was washed 4×100 ml with methylenechloride. The combined organic phases were dried over sodium sulfate,filtered and evaporated. The residue was recrystallized with a minimalamount of methylene chloride and a larger amount of ether. The yield was46.3 g (87%) of (R)-1-(5-hydroxyhexyl)-3,7-dimethyl xanthine m.p.105°-108° C., [α]D=-5.63, ca. 96% ee.

EXAMPLE 3

This example illustrates a process for preparing of(R)-1-(5-hydroxyhexyl)-3,7-dimethylxanthine (CT1501R) using DICHED as achiral director. The pinanediol chiral director described in example 2can be replaced with (1S,2S)-(1,2)-dicyclohexylethanediol (DICHED). Thischiral director is more easily recovered (95%) than the pinanediol.DICHED can be prepared according to a procedure described in Sharplesset al., J. Org. Chem. 57:2768, 1992. Dimethyl-4-bromobutylboronate (10.1g, 48.06 mmol), prepared as described above was mixed with 10.54 g (46.6mmol) of (S,S)-DICHED in 100 ml of ether. After 30 min, the ether wasremoved and the residue put through a short column of 10 g of silica geland eluted with petroleum ether/ether (9:1), yielding 17.8 g of the(S,S) DICHED analog 4-bromobutylboronate.

The homologation reaction was performed as described in example 2 withthe following amounts: 17.8 g of DICHED 4-bromobutylboronate; 6.11 g ofmethylene chloride; 41.2 ml of 1.4N n-butyllithium; 100 ml of THF and5.89 g of anhydrous zinc chloride.

The Grignard reaction was performed, as in example 2, with the followingamounts: DICHED (R)-1-chloro-5-bromopentylboronate, 20.04 g; 3.0Nmethylmagnesium bromide, 16.7 ml; 150 ml of THF. The oxidation of theDICHED boronate to give (R)-6-bromohexan-2-ol was performed bydissolving the DICHED 5-bromo-1-methylpentylboronate in THF (10 ml) in a50 ml flask. Water (10 ml) was added and the solution. The solution wascooled to 0° C. while stirring under argon. Sodium carbonate (16.5 ml ofa 3M solution) was added, followed by 8 ml of 30% hydrogen peroxide. Thesolution was filtered, 20 ml of pentane added, and filtered again. Theaqueous phase was separated and washed with pentane (2×10 ml). Theorganic phase was dried over sodium sulfate, filtered and the solventevaporated to give (R)-6-bromo-hexan-2-ol in a crude yield of 8.9 g.Vacuum distillation provided a yield of 7.1 g (84%) of pure materialwith a rotation of -13.89 [α]₅₄₉.

The (R)-bromoalcohol was added to theobromine. A mixture of theobromine(2.02 g, 11.2 mmol) was stirred in DMSO (30 ml), and 282 mg of sodiumhydride (11.8 mmol) was added. The reaction mixture was vigorouslystirred for 80 minutes. The bromoalcohol (2.03 g, 11.2 mmol) was addeddropwise and the stirring continued for 21 hr. DMSO was distilled offunder full pump vacuum. Water (100 ml) was added. The mixture wasextracted with 25% ethanol/methylene chloride (3×50 ml) and the combinedextracts were dried over magnesium sulfate and evaporated. The residuewas taken up in 20 ml of methylene chloride and 150 ml of ether wasadded. Beige crystals formed of(R)-1-(5-hydroxyhexyl)-3,7dimethylxanthine (1.5 g, 5.36 mmol, 47.8%yield). Another 500 mg of the product crystallized over the next 24 hrto give a total yield of 2 g (64% overall) with greater than 94% ee bychiral chromatography.

EXAMPLE 4

This example illustrates a process for synthesis of (R orS)-1-(6-hydroxyheptyl)-3,7-dimethyl-10 xanthine or (R orS)-7-hydroxyoctyl)-3,7-dimethylxanthine. These compounds were preparedusing the appropriate pinanediol and DICHED procedures described aboveby using as starting materials the longer chain bromo-olefins,5-bromo-1-pentene and 6-bromo-1-hexene, 15 respectively.

EXAMPLE 5

This example illustrates a mixed lymphocyte reaction of CT1501R and PTX.The mixed lymphocyte reaction shows a proliferative response of PBMC(peripheral blood mononuclear cells) to allogeneic stimulationdetermined in a two-way mixed lymphocyte reaction. Both CT1501R and PTXshowed activity in this immune modulating activity assay procedure asshown in FIG. 1.

EXAMPLE 6

This example shows the effects of CT1501R on inhibition of murine B-cellproliferation stimulated by anti-mu antibody crosslinked and/orinterleukin-4 (IL-4). FIG. 2 shows that CT1501R inhibited B-cellproliferation caused by the indicated proliferative signals.

FIG. 3 shows the effects of CT1501R inhibiting proliferation caused byConcanavalin A (ConA) and interleukin-1 alpha (IL-1α) or interleukin-2(IL-2). CT1501R was added to the cells at the doses indicated two hoursprior to activation with ConA and IL-1α or IL-2. CT1501R inhibitedthymocyte proliferation in a dose-response manner as is shown in FIG. 3.Background counts were less than 200 cpm.

FIG. 4 shows the effects of CT1501R2 and PTX on inhibition of smoothmuscle proliferation stimulated by PDGF (platelet derived growth factor)and IL-1. CT1501R and PTX were separately added to the cells two hoursprior to activation with PDGF and IL-1. Both drugs inhibited smoothmuscle cell proliferation at the higher doses tested as shown in FIG. 4with CT1501R being more active than PTX.

EXAMPLE 7

This example illustrates in vitro effects of CT1501R, includinginhibition of cytokine release and cellular adhesion. We determined theeffects of CT1501R on endotoxin, TNF-α or IL-1α-stimulated cytokinerelease and adhesion to activated human umbilical endothelial cells.

Murine peritoneal exudate cell macrophages (PEG) were isolated byperfusion of mouse peritoneum and plated into 96-well trays at 5×10⁵cells per well. Cells were stimulated with 5 μg/ml Salmonella abortusequi-derived endotoxin (LPS; Sigma Chemical Co., L-1887) or 50 ng/mlIL-1α (Genzyme; Cambridge, Mass.) with or without the addition ofCT1501R added to the cultures one hour prior to addition of thestimulus. At various times thereafter, supernatants were removed andlevels of either TNF-α or IL-1α were assayed using commercial 96-wellmicrotiter immunoassay kits (Genzyme; TNF; Endogen, Boston, Mass.;IL-1α) according to manufacturer's specifications. For the adhesionstudies, early passage human umbilical vein endothelial cells (HUVEC)were obtained from commercial suppliers (Clonetics, San Diego, Calif. orCell Systems, Seattle, Wash.) and cultured in defined, Hepes buffered,serum free medium (Cell Systems, cat 301-180) supplemented with acidicFGF (Cell Systems cat 401-111). 4000 cells were plated into each well ofa 96-well microtiter plate and allowed to incubate for 72 hrs at 37° C.The histiocytic leukemia cell line U937 was stimulated in RPMI 1640medium supplemented with 10% fetal calf serum. Either the HUVEC or U937cells were stimulated with either LPS, IL-1α or TNF-α for 12 hrs. Forthe adhesion assay, U937 cells were labeled with the fluorescentviability stain, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluoresceinacetoxymethyl ester (BCECF). Briefly, the cells were labeled with 10μg/ml BCECF in RPMI-1640 media plus 10% fetal calf serum for 30 min at37° C. The cells were wished once with full media and 50,000 cells/welladded to the HUVEC cultures. The samples were incubated at 37° C. for 30min, inverted and centrifuged for 10 min at 400×g. After addition of 100ml Hanks Balanced Salt Solution (HBSS), the microtiter plate was thenanalyzed on a Millipore Cytofluor fluorescent plate reader (excitation488 nm; emission 525 nm). Fluorescence of serial dilutions of cellsshowed a linear concentration relationship over wide ranges of cellconcentrations from 20 to 100,000 cells per well (data not shown). Allexperiments were repeated at least in duplicate with all resultsconfirming the findings of those shown.

CT1501R inhibited TNF-α release from LPS stimulated PEG cells. Cellswere pretreated with 250 mM CT1501R and supernatants assayed by ELISAfor released TNF-α as a function of time following LPS stimulation.CT1501R significantly inhibited TNF-α release at all time pointsmeasured from approximately 50% of LPS induced levels at 6-24 hrs toapproximately 70% at 48 hr. A dose response of the effects of CT1501R onreleased TNF is shown in FIG. 18. PEG cells were stimulated with LPS 1hour following treatment with various concentrations of CT1501R. Theconcentration for CT1501R 50% inhibition was approximately 30 μM. At 500μM the inhibitory effects of CT1501R were approximately 40% of themaximal LPS stimulation (1428 pg/ml).

LPS stimulates IL-1α release in peritoneal macrophages, increasing tolevels of 24 pg/ml by 12 hours following LPS stimulation, and 31 pg/mlby 48 hrs (FIG. 19). Addition of CT1501R significantly attenuated IL-1αrelease at 12-48 hrs, with a maximum inhibition of approximately 50% at24 hours. If the peritoneal macrophages were stimulated with 50 ng/mlIL-1α rather than LPS (FIG. 20), CT1501R also inhibited release of TNF-α(FIG. 20). Addition of CT1501R resulted in a 50%-70% inhibition ofinduced levels of TNF-α at the tested time points.

Cells of the immune lineage leave the blood by recognizing and bindingto vascular endothelial cells. Thereafter immune cells migrate betweenendothelial cells and surrounding tissue. Models of immune cell adhesionto endothelial cells and extravasation provide predictive models for invivo manipulation an inflammatory and an atherogenic response.Activation of endothelial cells with TNF-α, IL-1α or LPS up-regulatescertain adhesion molecules and increases their adhesiveness forlymphocytes, monocytes, eosinophils and neutrophils. The cell line U937was used to test the effects of CT1501R on inhibition of adhesion toactivated HUVEC. U937 cells express many of the phenotypiccharacteristics of monocytes including expression of the integrin VLA-4,which mediates monocyte attachment to endothelial cells via vascularadhesion molecule 1 (VCAM), a member of the immunoglobulin superfamilywhich is expressed on endothelial cells. HUVEC were treated overnightwith an activating agent selected from IL-1≢, TNF-α or LPS with orwithout the addition of 250 μM CT1501R, added 1 hour prior to additionof the activating agent. As shown in FIG. 21, there was no significanteffect on background attachment of U937 cells to HUVEC by the additionof CT1501R. However, there was a marked suppression in the relativeadhesiveness of the activated HUVEC pretreated with CT1501R. Thisinhibition by CT1501R was observed with stimulation of the HUVEC witheither TNF-α, IL-1-α or LPS.

Conversely, activating U937 cells with IL-1-α also increased theirrelative adhesiveness to non-stimulated HUVEC (FIG. 22). Pre-treatmentof U937 cells with 250 μM CT1501R effectively blocked the IL-1α mediatedincrease in adhesion to HUVEC to near background levels.

CT1501R inhibited LPS, TNF-α and IL-1α-mediated inflammatory signalingpathways and concomitant cellular responses in peritoneal macrophages,the human U937 histiocytic leukemia cell line, and in human umbilicalvein endothelial cells. CT1501R significantly decreased release of thepro-inflammatory cytokines TNF-α and IL-1α from peritoneal macrophagesstimulated with LPS. The IC50 for TNF-α inhibition using 10 μg/ml LPSstimulation was approximately 30 μM. CT1501R blocked TNF-α release fromIL-1α activated PEC. CT1501R inhibited the increase in adhesion of U937cells to TNF-α, IL-1α or LPS activated HUVEC. Finally, CT1501R inhibitedIL-1α-induced activation and increased adhesiveness of U937 cells tonon-stimulated HUVEC.

EXAMPLE 8

This example illustrates nude mouse hair studies involving topicalapplication of CT1501R. Six to eight week old, female, nu/nu mice fromCharles River Laboratories were housed at Biosupport Research SupportServices (Seattle, Wash.) in autoclaved micro isolator units withhyperchlorinated autoclaved water, irradiated rodent chow and kept undera laminar flow hood. Caging was changed weekly, and water was changedtwice weekly. The mice were acclimated for 5 days before beginning eachstudy.

The first topical formulation Was prepared by adding CT1501R to a heatedhydrophilic ointment (USP) at a 1% concentration, and allowed tosolidify.

Nude mice were painted twice daily for 16 days with the first topicalformulation of CT1501R on the left flank and another compound in thesame base on the right flank with sterile applicators. Mice were handledunder the laminar flow hood with applicator wearing face mask andsterile gloves. After 16 days, one mouse was sacrificed by cervicaldislocation and skin biopsies were taken of the treated areas of theshoulder/flank and from the non-treated area of the dorsal pelvis(rump). Specimens were placed in 10% buffered formalin solution.Biopsies were sent to a veterinary dermopathologist for histopathology.Six weeks following treatment, a second mouse was euthanatized andbiopsied the same as the first. Samples were sent for histopathology.

The results from the first experiment show that treated sections hadsignificantly more normal appearing hair follicles than the non treatedsections. Numerous hair shafts were seen exiting the follicles in thetreated sections vs. none in the non treated sections for both CT1501Rand the other compound.

In a second experiment, topical application of CT1501R was performedalong with a commercial topical minoxidil preparation (Rogaine®, Upjohn)that is approved for a hair growth indication. Five to six week old,female, nu/nu mice from Bantin and Kingman Universal were housed atBiosupport Research Support Services (Seattle, Wash.) in autoclavedmicro isolator units with hyper chlorinated autoclaved water, irradiatedrodent chow and kept under a laminar flow hood. Caging was changedweekly, and water was changed twice weekly. Mice were acclimated for 7days before beginning study.

CT1501R, and two other compounds were prepared in a topical formulationof 60% ETOH, 10% water and 30% PEG along with vehicle only. Minoxidilwas purchased as the commercial preparation Rogaine® from a localpharmacy. Rogaine® is sold in the same formulation base. Mice wereidentified by unique tail markings specific for each test article. Twomice per group were treated with CT1501R and another compound. One mousewas treated with each of vehicle and minoxidil. The mice were handledunder the laminar flow hood with the applicator wearing face mask andsterile gloves. Each mouse was treated twice daily with a separatesterile applicator to avoid contamination of the solutions or the mice.All mice were applied a strip of appropriate test article along thecenter line of the back from the base of the skull to the base of thetail, approximately 1.5 cm wide. After 34 days all mice wereeuthanatized by overdose of halothane anesthesia. Skin biopsies weretaken from each of the 7 mice over the nape of the neck between thescapulas (approximate size was 1×1.5 cm). All specimens were blotted ongauze to remove blood and fluids, affixed to a cut piece of woodentongue depressor, and placed specimen side down in separately markedcontainers with 10% buffered formalin. Specimens were shipped to aveterinary dermatopathologist for qualitative histopathologicaldeterminations.

Subjective examination revealed that the vehicle treated mouse (control)had the least well developed hair follicles. A fair response totreatment was evident in the minoxidil treated mouse. CT1501R and theother compound treated mice had the greatest concentration of hairfollicles. Further, visual inspection of the mice revealed that theCT1501R mice were the most "hairy" of a group consisting of CT1501R,minoxidil and control. Therefore, CT1501R is at least as good atpromoting hair growth in this model as minoxidil. Accordingly, a topicalformulation of 1-4% CT15001R (by weight) is an effective therapeuticcomposition for treating baldness and promoting hair growth.

EXAMPLE 9

This example illustrates the effect of CT1501R on survival in mice givena lethal dose of endotoxin. We determined if CT1501R protects againstendotoxin induced lethality in a murine model. Septic shock was modeledby endotoxin injection of 6-8 week old female Balb/c mice similar topreviously published reports (Ashkenazi et al. Proc. Natl. Acad. Sci.U.S.A. 88: 10535-10539, 1991.) under protocols approved by the AnimalUse Committee of the Biomembrane Institute, Seattle, Wash. Animals wereinjected intravaneously (i.v.) with an approximate LD₁₀₀ dose (10 μg/g)of Salmonella abortus equi-derived endotoxin (Sigma Chemical Co.,L-1887) in phosphate buffered saline (PBS). CT1501R at a dose of 100μg/kg was injected intraperitoneally (IP) 3 times per day (100ml/injection). Control mice were injected at the same times with asimilar volume of vehicle control (PBS). Survival was followed for atleast 72 hours.

For ELISA measurements of cytokine levels in plasma, blood was collectedby retro-orbital or cardiac puncture of anesthetized mice, immediatelycentrifuged and the EDTA plasma stored at -70° C. Particulate freeplasma was thawed on ice and cytokine levels determined utilizingcommercial ELISA assays with normal mouse plasma used to generatestandard curves. Murine tumor necrosis factor-α and interleukin-1α kitswere purchased from Genzyme (Cambridge, Mass.) and Endogen (Boston,Mass.), respectively. Each data point was an average of two ELISAmeasurements made from EDTA serum pooled from three mice. The datasummarized in Table 3 were compiled from two independent experiments;data from Table 4 and 5 were from single experiments of three mice perdata point.

Ten mice were treated each with PBS alone or CT1501R alone on the sameschedule as the experimental mice. There were no adverse effects notedand survival was 100% throughout the course of the experiment (data notshown). Endotoxin survival data were summarized from a total of six (6)independent experiments. Summary Table 1 details the results of each ofthe six experiments. Cumulative percent survival is given in Table 2 andplotted as FIG. 5. In FIG. 5, each time point represents a minimum ofthree (3) experiments comprising at least twenty (20) mice per group. Aprobability analysis of the survival data using a Fisher's Exact OneTailed Test is given as Table 3. Significant, protection was conferredif CT1501R was administered immediately after the LPS treatment. Thecumulative percent survival at 72 hours post LPS treatment was 60%compared to 7% for the LPS-only treated animals (p=<0.0005; Table 3).

The effect of delaying the time of administration of CT1501R followingthe LPS treatment is also shown in FIG. 5. Animals Were treated with LPSand given CT1501R either two or four hours after the LPS treatment.Again, CT1501R conferred significant protection. Survival of the CT1501Rtreated mice was 55% at 2 hrs and 37% at 4 hrs compared to 7% for theLPS-only treated mice (p=0.0005 and p=0.001; respectively; Table 3).

Levels of TNF-α, IL-1α and IL-6 were measured in the plasma of mice as afunction of time following treatment with S. abortus endotoxin. Thesedata were compared to animals treated with endotoxin followedimmediately with a single i.p. injection of CT1501R. TNF-α levels peakedwithin 1 hour of treatment of endotoxin (Table 4 and FIG. 6). Treatmentof the mice with CT1501R decreased the levels of TNF-α in the EDTAplasma of endotoxin treated mice at all time points measured. Inparticular, peak levels of TNF-α at 0.5 and 1 hour post endotoxin weredecreased 2.5 and 2.6 fold respectively in the CT1501R treated mice.

Plasma levels of IL-1α were also decreased by treatment with CT1501Rimmediately following the endotoxin (Table 5; FIG. 7). In particular,peak levels observed at 6, 12 and 18 hours post endotoxin were decreased1.2, 4.3 and 3.2-fold, respectively. Finally, IL-6 measurements weremade in a similar manner (Table 6, FIG. 8). Peak plasma levels at 3, 6and 12 hours were also decreased in the CT1501R treated animals (1.7,2.0 and 4.1-fold decrease, respectively).

CT1501R significantly enhanced survival in mice that received a dose ofendotoxin that was lethal to 41 of 44 mice. Survival was improvedcompared to control when CT1501R was administered simultaneous withendotoxin or after 2 or 4 hours following endotoxin treatment.Administration of a single dose of CT1501R immediately following theendotoxin significantly decreased peak plasma levels of TNF-α, IL-1α andIL-6.

                  TABLE 1    ______________________________________    Survival data from six independent mouse sepsis experiments.            Time of                   Hours Post LPS            CT-1501R                   (Number Surviving/Total)    Expt. #   Post LPS 24 Hr      48 Hr                                       72 Hr    ______________________________________    1         LPS only 2/4        0/4  0/4              t = 0 Hr 4/5        4/5  4/5              t = 2 Hr 4/5        3/5  3/5    2         LPS only 0/5        0/5  0/5              t = 0 Hr 5/5        5/5  5/5              t = 2 Hr 5/5        5/5  5/5    3         LPS only 1/5        1/5  1/5              t = 4 Hr 5/5        3/5  3/5    4         LPS only 1/10       1/10 1/10              t = 4 Hr 7/10       5/10 5/10    5         LPS only 0/10       0/10 0/10              t = 0 Hr 9/10       7/10 3/10              t = 2 Hr 5/10       3/10 3/10              t = 4 Hr 7/10       4/10 2/10    6         LPS only 3/10       1/10 1/10              t = 4 Hr 3/10       3/10 3/10    ______________________________________

                  TABLE 2    ______________________________________    Cumulative mouse survival from data derived from Table 1.            Time of                   Hours Post LPS    No.       CT-1501R (Number Surviving/Total)    Expts.    Post LPS 24 Hr      48 Hr                                       72 Hr    ______________________________________    n = 6     LPS only  7/44       3/44                                        3/44    n = 3     t = 0 Hr 18/20      16/20                                       12/20    n = 3     t = 2 Hr 14/20      11/20                                       11/20    n = 4     t = 4 Hr 22/35      15/35                                       13/35    ______________________________________

                  TABLE 3    ______________________________________    Fisher's Exact P Values (one tailed)    for the mouse sepsis survival data in Table 2.            Time of                   P Value(Hours Post LPS)    No. Expts.              CT-1501R 24 Hr     48 Hr  72 Hr    ______________________________________    n = 6     LPS only    n = 3     t = 0 Hr <0.0005   <0.0005                                        <0.0005    n = 3     t = 2 Hr <0.0005   0.0005 0.0005    n = 4     t = 4 Hr <0.0005   0.0002 0.001    ______________________________________

                  TABLE 4    ______________________________________    Averaged Data From Mouse Plasma    TNF-α ELISA Measurements    Hours Post    Plasma TNF (pg/ml)    LPS           LPS Only LPS + CT-1501R    ______________________________________    0              0        0    0.17           8        6    0.5           990      390    1             4480     1728    2             1585     940    4             655      400    6             545      350    ______________________________________

                  TABLE 5    ______________________________________    Data From Mouse Plasma IL-1α ELISA Measurements    Hours Post    Plasma IL-1 (pg/ml)    LPS           LPS Only LPS + CT-1501R    ______________________________________    0             0        0    0.17          0        0    0.5           0.01     22    1             2.25     10.8    3             41.3     33.2    6             166      133    12            139      32.3    18            150      46.7    24            16.6     43.5    36            all dead 18.8    48            all dead 0    ______________________________________

                  TABLE 6    ______________________________________    Data From Mouse Plasma IL-6 ELISA Measurements.    Hours Post    Plasma IL-6 (ng/ml)    LPS           LPS Only LPS + CT-1501R    ______________________________________    0             0        0    0.17          0.2      0.14    0.5           3.86     1    1             24       31.8    3             317      183    6             247      121    12            119      29.6    18            59       19.8    24            31       25.6    36            0        1.64    48            2        0.4    ______________________________________

EXAMPLE 10

This example illustrates the effect of CT1501R on 5-fluorouracil (5-FU)induced bone marrow suppression in mice. We determined if CT1501Rinfluenced the time required for hematopoietic reconstitution followingcytotoxic chemotherapy in a murine model. Female Balb/C mice (VAF,Charles River Laboratories, 6-8 wks of age approximately 17.3-18.5 g)were treated with 5-FU at a dose of 200 mg/kg intraperitoneally (i.p.)in experiment 1 or 190 μg/kg in experiment 2. CT1501R or vehicle controlwas given at a dose of 100 μg/kg i.p. bid starting 1 day prior to 5-FUand was continued until the last mice were sacrificed on day 13 inexperiment 1 and day 15 in experiment 2. Controls included mice treatedwith CT1501R or vehicle without 5-FU. Mice (4 per group) were sacrificedstarting 2 days after 5-FU by cardiac puncture under halothaneanesthesia followed by cervical dislocation and had total white bloodcell counts and differential counts performed. Platelet counts wereperformed using phase-contrast microscopy in duplicate. Femurs wereharvested and the number of granulocyte-macrophage colony forming cells(CFU-GM) per femur were measured using a standard assay (Terry FoxLaboratories, Vancouver, British Columbia) using pokeweed mitogen spleenconditioned medium as the growth factor. Each femur was platedseparately in triplicate and the mean and means for each experimentalpoint was calculated. Standard deviations used for statistical analysiswere deviations of the mean number of colonies measured for each femur.

The mice treated with vehicle control or CT1501R had no apparent adverseeffects. The mice treated with the vehicle control alone had a rise inabsolute neutrophil count (ANC) and white blood cell counts (WBC) withtime which was not seen in the CT1501R treated mice. These differenceswere significantly different from day 0 values on days 4 and 8 (p=0.012and p=0.016, respectively; two tailed student T test). When compared tomice receiving CT1501R, the control treated mice had significantlyhigher white blood cell counts on day 4 (p=0.009) and significantlyhigher neutrophil counts on day 12 (p=0.028). Control treated mice had asignificant rise in granulocyte count on day 12 compared to the value onday 0 (p=0.028). The CT1501R treated mice's WBC and ANC remained within1 SD of control values.

The WBC's of 5-FU treated mice were significantly lower in vehicletreated controls than in CT1501R treated mice on days 6 and 10 (p=0.019and 0.036, respectively) (FIG. 10). On differential blood counts allcells had the morphology of lymphocytes in both groups on days 6, and 8.Some monocytes and rare granulocytes were noted in the CT1501R treatedanimals on day 10. On day 13, CT1501R had a mean ±SD ANC of 440±17/mm³while the control mice had 180±10/mm³ (P=0.05) (FIG. 10).

An estimate of recovery of hematopoietic progenitor cells is provided bymeasurement of the number of CFU-GM/femur. Following 5-FU treatment bothvehicle control and CT1501R treated mice had suppression of CFU-GM tonear unmeasurable levels until day 8 when recovery began (FIG. 13). Byday 13, there was significant divergence. CT1501R treated mice hadsignificantly more CFU-GM/femur on day 13 than either the vehiclecontrol animals (p=0.024) and animals sacrificed before any treatment onday 0 (p=0.05) indicating that there was an overshoot of progenitorrecovery.

Experiment 2 was similar to experiment 1 with the following exceptions:(1) the dose of 5-FU was decreased to 185 mg/kg; (2) daily blood drawswere performed between days 10 and 15; and (3) platelet counts wereperformed. Results from experiment 2 are displayed graphically in FIGS.10-13. As in the first experiment, mice treated with CT1501R hadsignificantly higher WBC's (FIG. 10) Neutrophil recovery Was acceleratedin the CT1501R treated mice (FIG. 12). The platelet count nadir and rateof recovery were also increased compared to vehicle control animals(FIG. 11) The number of cells per femur was increased duringhematopoietic recovery compared to control animals as was the number ofCFU-GM/femur (FIG. 13).

In mice that received a highly marrow suppressive dose of 5-FU, CT1501Rtreatment increased the rate of rise in neutrophil counts and the rateof marrow repopulation with committed myeloid progenitor cells. CT1501Rinhibited 5-FU induced suppression of the total WBC at each time pointmeasured. However, until day 10, the increase was in cells with themorphologic appearance of small lymphocytes. On day 13 in experiment 1and on day 10 in experiment 2, the neutrophils in mice treated withCT1501R became significantly higher than in vehicle control treatedmice; The stimulation of hematopoietic recovery by CT1501R also affectedthe megakaryocyte lineage. Test animals had a significantly higherplatelet nadir than control animals and had a more rapid rise inplatelet count and a greater overshoot than did vehicle controls.

Stimulation of hematopoiesis was also evidenced by both the return inmarrow cellularity and the quantification of marrow progenitor cells.CT1501R treated mice had an approximately two-fold increase in thenumber of CFU-GM/femur during hematopoietic recovery compared to controlanimals.

In animals that did not receive 5-FU, treatment with CT1501R preventedboth the rise in ANC and rise in CFU-GM/femur associated with thevehicle control. All CT1501R treated animals maintained ANC's within onestandard deviation of non-injected controls. It appears that CT 1501Rprevented the stress-induced and probably cytokine-mediated response totwice daily i.p. injections.

Overall, these data support a method of using CT1501R and the inventivecompounds to accelerate the reconstitution of hematopoiesis followingcytotoxic drugs or cytoreductive therapies.

We claim:
 1. A method for treating or preventing hematopoietic or organtoxicity caused by cytoreductive therapies to a patient in need of suchtreatment, comprising administering an effective amount of a compoundwherein the compound has a formula: ##STR7## wherein: R₁ is asubstantially pure resolved R enantiomer ω-1, secondaryalcohol-substituted alkyl (C₅₋₈) group; andR₂ and R₃ are independentlyhydrogen atom or an alkyl (C₁₋₁₂) or alkoxyl (C₁₋₁₂), or apharmaceutical composition thereof.
 2. The method of claim 1 wherein thecompound is 1-(R)-(5-hydroxyhexyl)-3,7-dimethylxanthine.
 3. The methodof claim 1 wherein the hematopoietic toxicity is in the form ofneutropenia, thromobcytopenia, anemia, or combinations thereof.
 4. Themethod of claim 1 wherein the organ toxicity is gastrointestinalmucositis.